Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus.

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G2/M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell.IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells.

Single-cell analysis reveals the relevance of foot-and-mouth disease virus persistence to emopamil-binding protein gene expression in host cells.

Foot-and-mouth disease virus (FMDV) infects host cells in either an acute or persistent manner. In this study, we examined the relevance of the establishment of FMDV persistence to the expression of the emopamil-binding protein (EBP) gene in 231 individual persistently infected baby hamster kidney (BHK-21) cells after passages 28, 38, and 68 (PI28, PI38, and PI68). At PI28, the stage at which persistent infection of FDMV becomes unstable, the percentage of cells carrying FMDV was 66.7%, while 80.2% of cells were EBP positive. Additionally, in 55.6% of the EBP-positive cells at PI28, EBP expression was upregulated approximately 149.9% compared to uninfected BHK-21 cells. This was the highest expression level among all cell passages measured. Interestingly, in a parallel experiment, the average EBP expression level in the whole cell population at PI28 was only slightly higher (108.2%) than that in uninfected BHK-21 cells. At PI38, 98.7% of the cells were positive for FMDV 3D (an RNA-dependent RNA polymerase enzyme gene), and its maximum expression level observed at this passage. The expression level of EBP in 78.2% of the total cells, however, was reduced significantly. At PI68, 95.8% of the cells were 3D positive, and the expression of both the EBP and 3D genes were at the lowest levels of all the passages. Our studies using single cells yielded data that are otherwise inaccessible a using whole cell population. These results suggest that the establishment of persistent infection by FMDV is a dynamic process that results from the continuous adaptation and coevolution of viruses and cells to reach an equilibrium.

Immunogenicity analysis of the E. coli expressed structural protein VP1 of persistent infection foot-and-mouth disease virus.

The persistent infection of FMDV in cloven hoofed animals has made the epidemic prevention and control more difficult. VP1 is the main immunogenic protein and first candidate of vaccine development for FMDV prevention. However, the mutation of VP1 in host cell with persistent infection FMDV (PI-FMDV) caused the change of its immunogenicity. Hence, it is imperative to establish the expression system for VP1 of PI-FMDV (PI-VP1) and re-evaluate its immunogenicity. In this study, the PI-VP1 with His-tag was cloned into pET-28a vector. PI-VP1 protein was expressed and purified in E. coli, and further the antiserum of immunized mice was analyzed. Results showed that purified PI-VP1 protein produced a good humoral and cellular immune response after immunizing mice. Furthermore, our study showed that the antiserum could not only neutralize PI-FMDV, but also prevent the adsorption of WT-FMDV. In summarize, our work provides valuable implications for the FMDV vaccines and therapeutics development.

Foot-and-mouth disease virus VP1 promotes viral replication by regulating the expression of chemokines and GBP1.

Foot-and-mouth disease virus (FMDV) is an acute, highly contagious, and economically destructive pathogen of vesicular disease that affects domestic and wild cloven-hoofed animals. The FMDV VP1 protein is an important part of the nucleocapsid and plays a significant role during FMDV infection. However, the signal pathways mediated by VP1 in the life cycle of FMDV and the related mechanisms are not yet fully understood. Here, we performed RNA-seq to compare gene expression profiles between pCAGGS-HA-VP1 transfected PK-15 cells and pCAGGS-HA (empty vector) transfected PK-15 cells. The results showed 5,571 genes with significantly different expression levels, of which 2,981 were up-regulated and 2,590 were down-regulated. GO enrichment analysis showed that 51 GO terms were significantly enriched in cell components including protein complex, membrane and organelle part. KEGG enrichment analysis showed 11 KEGG pathways were significantly enriched which were mainly related to the immune system, infectious viral disease, and signal transduction. Among the up-regulated genes, the chemokines such as CCL5, CXCL8, and CXCL10 in turn promoted FMDV replication. In contrast, GBP1, an interferon-stimulated gene that was suppressed by VP1 and FMDV, could effectively inhibit FMDV replication. Our research provides a comprehensive overview of the response of host cells to VP1 protein and a basis for further research to understand the roles of VP1 in FMDV infection including the genes involved in FMDV replication.

Single-Cell Analysis of Foot-and-Mouth Disease Virus.

With the rapid development of single-cell technologies, the mechanisms underlying viral infections and the interactions between hosts and viruses are starting to be explored at the single-cell level. The foot-and-mouth-disease (FMD) virus (FMDV) causes an acute and persistent infection that can result in the break-out of FMD, which can have serious effects on animal husbandry. Single-cell techniques have emerged as powerful approaches to analyze virus infection at the resolution of individual cells. In this review, the existing single-cell studies examining FMDV will be systematically summarized, and the central themes of these studies will be presented.

Transferrin-Modified Nanoliposome Codelivery Strategies for Enhancing the Cancer Therapy.

Chemotherapy remains one of the most effective treatments for many cancers in a clinic. At present, various targets have been used to modify the PEGylated liposomes for doxorubicin (Dox) delivery, but the antitumor effect of Dox is not satisfactory. Therefore, combination chemotherapeutics has been considered as a promising method to improve tumor treatment. These years, RAF/MEK/ERK-mediated cell signaling pathway has been discovered to inhibit the growth of tumors. Thus, Sorafenib tosylate (Sor) was used in this study, which directly inhibited tumor cell proliferation through blocking RAF/MEK/ERK-mediated cell signaling pathway and indirectly inhibited tumor cell growth through blocking angiogenesis by VEGFR and PDGF. In this article, we develop a "combination delivery system" to deliver the hydrophobic drug (Sor) in phospholipid bilayer and hydrophilic drug (Dox) in inner cores for enhancing the antitumor effect. Moreover, in vitro experiments verified whether the physicochemical properties of carriers were stable and transferrin-modified liposomes displayed the highest uptake. The results of in vivo experiments showed that the codelivery system inhibited the tumor growth more effectively than monotherapy. Overall, this combination delivery system for delivering the hydrophobic and hydrophilic drugs simultaneously may offer a novel strategy for breast cancer treatment and provide a reference for the possibility of clinical usage.

Comparative Transcriptome Analysis Reveals Different Host Cell Responses to Acute and Persistent Foot-and-Mouth Disease Virus Infection.

Foot-and-mouth disease virus (FMDV) rapidly causes cytopathic effects in susceptible cells. Incomplete viral clearance during the acute infection leads to persistent infection. The relationship between host gene expression and the persistent infection remains unclear. In this study, we analyzed the transcriptome profiles of BHK-21 cells acutely and persistently infected with FMDV to identify differences in gene expression. GO and KEGG enrichment analyses showed that the 8,378 differentially expressed genes were significantly enriched in categories including metabolism, biosynthesis, ribosome function, and endocytosis. In persistently infected BHK-21 cells, ribosome- and translation-related genes were significantly down-regulated. There were more differentially expressed immune-related genes during persistent infection than during acute infection. Two hundred and seventy-four genes were differentially expressed in both acutely and persistently infected BHK-21 cells. Among these genes, heat shock protein family B member 1 (Hspb1) knockdown significantly inhibited FMDV replication. Our research provides a basis for further research to understand the mechanisms of persistent FMDV infection including the genes involved in FMDV replication.

Cellular response to persistent foot-and-mouth disease virus infection is linked to specific types of alterations in the host cell transcriptome.

Food-and-mouth disease virus (FMDV) is a highly contagious virus that seriously threatens the development of animal husbandry. Although persistent FMDV infection can dramatically worsen the situation, the mechanisms involved in persistent FMDV infection remain unclear. In the present study, we identified the presence of evolved cells in the persistently FMDV-infected cell line. These cells exhibited resistance to the parent FMDV and re-established persistent infection when infected with FMDV-Op (virus supernatant of persistent infection cell lines), emphasizing the decisive role of evolved host cells in the establishment of persistent FMDV infection. Using RNA-seq, we identified the gene expression profiles of these evolved host cells. In total, 4,686 genes were differentially expressed in evolved cells compared with normal cells, with these genes being involved in metabolic processes, cell cycle, and cellular protein catabolic processes. In addition, 1,229 alternative splicing events, especially skipped exon events, were induced in evolved cells. Moreover, evolved cells exhibited a stronger immune defensive response and weaker MAPK signal response than normal cells. This comprehensive transcriptome analysis of evolved host cells lays the foundation for further investigations of the molecular mechanisms of persistent FMDV infection and screening for genes resistant to FMDV infection.

[Expression of liposome-mediated PEDF-GFP gene in the retina of the BN rats through different gene delivery route].

OBJECTIVE: Transducing PEDF-GFP plasmid to the retina of the BN rats with cationic liposome through different gene delivery route, then observe the expression and location of the PEDF-GFP. METHODS: PEDF-GFP plasmid with cationic liposome was delivered to the retina of the BN rat through subretinal injection and intravitreal injection. The expression of GFP was observed under fluorescence microscope, and the mRNA of PEDF gene was detected by RT-PCR. RESULTS: Green fluorescence was emitted from the total retina include RPE cell under fluorescent microscope after 24 h in two gene delivery route, The fluorescence intensity was stronger with time changing. Gradual fluorescence increase in the retina and RPE cells occurred and lasted 4 weeks. The expression of PEDF mRNA was also detected by RT-PCR after 24 h, and maintained stable 4 weeks after infection. CONCLUSIONS: Cationic liposome can mediate PEDF-GFP gene into the retina of the BN rat effectively; subretinal injection and intravitreal injection both are effective gene delivery route; and their stable expression can maintain 4 weeks after transfection.

Systematic evaluation of multifunctional paclitaxel-loaded polymeric mixed micelles as a potential anticancer remedy to overcome multidrug resistance.

Multidrug resistance (MDR) of tumor cells is becoming the main reason for the failure of chemotherapy and P-glycoprotein (P-gp) mediated drug efflux has demonstrated to be the key factor for MDR. To address this issue, a novel pH-responsive mixed micelles drug delivery system composed of dextran-g-poly(lactide-co-glycolide)-g-histidine (HDP) and folate acid-D-α-tocopheryl polyethylene glycol 2000 (FA-TPGS2K) copolymers has been designed for the delivery of antitumor agent, paclitaxel (PTX) via FA-receptor mediated cell endocytosis, into PTX-resistant breast cancer MCF-7 cells (MCF-7/PTX). PTX-loaded FA-TPGS2K/HDP mixed micelles were characterized to have a small size distribution, high loading content and excellent pH-responsive drug release profiles. Compared with HDP micelles, FA-TPGS2K/HDP mixed micelles showed a higher cytotoxicity against MCF-7 and MCF-7/PTX cells due to the synergistic effect of FA-receptor mediated cell endocytosis, pH-responsive drug release and TPGS mediated P-gp inhibition. P-gp expression level, ATP content and mitochondrial membrane potential change have been measured, the results indicated blank FA-TPGS2K/HDP mixed micelles could inhibit the P-gp activity by reducing the mitochondrial membrane potential and depleting ATP content but not down-regulating the P-gp expression. In vivo antitumor activities demonstrated FA-TPGS2K/HDP mixed micelles could reach higher antitumor activity compared with HDP micelles for MCF-7/PTX tumor cells. Histological assay also indicated that FA-TPGS2K/HDP mixed micelles showed strongly apoptosis inducing effect, anti-proliferation effect and anti-angiogenesis effect. All these evidences demonstrated this pH-sensitive FA-TPGS2K/HDP micelle-based drug delivery system is a promising approach for overcoming MDR. STATEMENT OF SIGNIFICANCE: In this work, a novel FA-TPGS2K copolymer has been synthesized and used it to construct mixed micelles with HDP copolymer to overcome MDR effect. Furthermore, a series in vitro and in vivo evaluations have been made, which supported enough evidences for the efficient delivery of antitumor drug to MDR cells.

Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis.

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.

Observation of a system of linear loops formed by re-growing hairs on rat skin.

This paper details linear hair re-growth patterns observed in rats. Adult rats were shaved and observed. The first wave of hair re-growth did not distribute everywhere, but along specific craniocaudally-oriented lines. The hair-lines were 2-15 mm wide and ran from the head, through the torso to the limbs, and were symmetrical along the left and right sides of the body. The symmetric hair-lines from both sides of the body converged around the mouth, nose, and at the pubic region or ventral midline to form a system of hair-loop-lines (HLLs). The loops can be differentiated into four main patterns. The Dorsal Loop and the Lateral Dorsal Loop run along the dorsum and hindlimb. The Ventral Loop and Lateral Ventral Loop travel along the thorax, abdomen, and forelimb. These hair-lines coincide with our previously observed sympathetic-substance lines (SSLs) in the rat's skin. Histological observation indicates that rat hair follicles along the hair-lines were at anagen phase. The catecholamine histofluorescent check showed abundant sympathetic nerve fibers beneath the hair-lines. After the rats' hairs were dyed, and selected portions shaved, re-growth was only observed on the shaved portions, indicating that the linear hair growth closely correlated with the shaving. Lastly we examine the cause of the preferential re-growth and briefly discuss the purpose and physiological role of the HLL.